Symbiodiniaceae

Sawelew, Ludovic, Nuccio, Christopher, Foord, Colin, Lorquin, Jean & Perez, Yvan, 2022, Symbiodiniaceae diversity and characterization of palytoxin in various zoantharians (Anthozoa, Hexacorallia), Organisms Diversity & Evolution (New York, N. Y.) 22 (3), pp. 555-576 : 569-572

Symbiodiniaceae diversity

According to previous studies, different Zoantharia species may have different patterns of association with Symbiodiniaceae ( Reimer et al., 2013) . In Zoanthus samples, our results highlighted a correlation between the geographical origin of the colony and the Symbiodiniaceae taxon. Indo-Pacific species predominantly hosted Cladocopium ( Z. kuroshio , 3/3; Z. sansibaricus , 4/5) and Durusdinium ( Z. gigantus , 1/1) whereas most species from the Caribbean-Atlantic basin hosted Symbiodinium ( Z. solanderi , 5/5; Z. aff. pulchellus , 2/3; Z. sociatus , 1/1). These findings are consistent with previous studies that identified Symbiodiniaceae belonging to the genus Cladocopium in Indo-Pacific Zoanthus species sampled in Japanese coastal waters ( Reimer & Todd, 2009; Reimer et al., 2013) while the genus Symbiodinium was predominant in colonies collected in coastal waters off Cape Verde and Brazil ( Reimer et al., 2010; Fontenele Rabelo et al., 2014; López et al., 2019). However, all Zoanthus specimens, including Z. pulchellus , Z. aff. pulchellus , and Z. solanderi , collected in the Canary Islands hosted Cladocopium ( López et al., 2019) . Cladocopium is considered an Indo-Pacific generalist, known from multiple hosts and environment ( Reimer et al., 2006c). According to Finney et al. (2010), who analysed the Symbiodiniaceae diversity of 45 genera of Cnidaria, symbiont specificity in the Caribbean is higher than that observed in the Indo-Pacific where Cladocopium dominates in many coral communities. This could be influenced by geographical isolation and habitat depth correlated with changes in ambient irradiance. Indeed, it has been shown that stress tolerant Symbiodinium thoa ) in this species. Similarly, Béress et al. (1983) were able to find 30 µg/g in wet Palythoa caribaeorum (Duchassaing and Michelotti, 1860) . In contrast to an earlier report ( Gleibs et al., 1995) and according to our results and the study of Arakate et al. (2016), PLTX or PLTX-like compounds were only detected in Palythoa colonies but not in other zoantharians such as Zoanthus or Terrazoanthus . Scale bars = 1 cm

and Durusdinium perform well at high irradiance and high temperature respectively and can increase bleaching resistance of the host ( LaJeunesse et al., 2009; Silverstein et al., 2015; Wang et al., 2012). In our study, the predominance of Symbiodinium spp. in Z. solanderi , Z. sociatus , and Z. aff. pulchellus from the Caribbean might suggest an adaptation to the environment in relation to abiotic stress tolerance. A similar strategy has been described in several Indo-Pacific zoantharian species collected in the Persian Gulf ( Symbiodinium spp. predominant) which was also interpreted as an adaptation to isolated regions characterized by an extreme physical environment ( Koupaei et al., 2016).

All Palythoa species analysed in this study were associated with Symbiodiniaceae belonging to the generalist genus Cladocopium . This same genus has been observed in all samples of P. tuberculosa and P. mutuki harvested in Japanese waters ( Mizuyama et al., 2020; Reimer et al., 2006c) as well as in colonies of the Penghu Islands of Taiwan ( Reimer et al., 2013). Similarly, all specimens of P. aff. clavata , P. grandiflora , and P. caribaeorum examined from the Canary and Cape Verde Archipelagos hosted Cladocopium , regardless of location or depth ( López et al., 2019). However, previous studies in Palythoa species revealed that their Symbiodiniaceae diversity is somewhat flexible being associated either with Cladocopium or Durusdinium , notably for Palythoa caesia Dana, 1846 from the Indian Ocean ( Burnett, 2002), P. tuberculosa and P. mutuki in Singapore waters ( Reimer & Todd, 2009) and in the northern Persian Gulf ( Koupaei et al., 2016) and P. tuberculosa in the Red sea ( Reimer et al., 2017a) and the South China Sea off the Vietnam coasts ( Sikorskaya et al., 2021). Our results, at least for the genus Zoanthus , support the idea that Symbiodiniaceae diversity is influenced by regional distribution and ecological specialisation, as previously suggested ( Mizuyama et al., 2020; Reimer et al., 2017b; Wee et al., 2020).

Interspecific and intraspecific toxicity variability in Zoantharia

One of the goals of this study was to use the evolutionary relationships revealed by molecular phylogenetic analyses, as well as the comparative analysis of PLTX contents, to predict zoantharian toxicity in a phylogenetic context and investigate the relation between toxicity and Symbiodiniaceae strains. To date, only one study of the relation between toxicity and phylogeny in Zoantharia has been conducted, with a reduced-scale taxonomic sampling ( Deeds et al., 2011). To assess the potential exposure of PLTX to marine aquarium hobbyists, specimens were identified through genetic analysis of 16S and COI markers. They investigated 16 specimens and tested the toxicity for the first time. They found four toxic specimens closely related to P. heliodiscus containing PLTX and PLTX-like compounds (range 0.5–3.5 mg /g wet zoantharian). Hamade et al. (2015) reported 7.3 mg and 6.2 mg /g wet zoantharian of PLTX in two specimens consistent with P. heliodiscus . These authors did not identified another PLTX analogue in these highly toxic specimens.

In the present study, none of the Zoanthus and Terrazoanthus tested contained PLTX or PLTX-like compounds but we observed toxicity variations in Palythoa . Specimens belonging to the heliodiscus complex were highly toxic which is consistent with the earlier findings of Deeds et al. (2011). Our results also demonstrated, for the first time, the presence of PLTX molecules in P. aff. clavata (Z26, Z27), an undescribed species from the Atlantic Ocean. Palythoa sp. Z05, another undescribed species from Indo-Pacific of the clavata complex, contains one of the highest amount of PLTX ever found in a zoantharian (2.22 ± 0.410 mg /g wet zoantharian). The mean recorded value of 0.22% (w/w) PLTX is high compared to the values found in the literature (see Table 1) and is eight times more than the first value of 0.027% (w/w) recorded by Moore and Scheuer (1971) from Palythoa toxica (Walsh & Bowers, 1971) . Fraga et al. (2017) demonstrated the presence of PLTX, 42-OH-PLTX, and six minor PLTX-like molecules in Palythoa canariensis Haddon and Duerden, 1896 , a species which has been considered a junior synonym to P. aff. clavata based on molecular data ( López et al., 2019). The PLTX content in P. canariensis was estimated at 0.27 mg /g of lyophilized zoantharian using UPLC-IT-TOF–MS. In the sample of P. heliodiscus (Z07), we identified a mixture of bishomoPLTX and PLTX. Although the concentration was high and corresponded to 0.16% (w/w), it was lower than the deoxyPLTX concentration of 0.35% (w/w) measured in the same species by Deeds et al. (2011) who also found a maximum of 1.164 mg /g wet zoantharian of PLTX corresponding to 0.12% (w/w). This result is very close to the concentrations of PLTX measured in both the purple (Z27, 0.13%) and the green (Z26, 0.11%) morphotypes of P. aff. clavata .

The PLTX molecule identified in Palythoa sp. Z05, P. aff. clavata (Z26, Z27) and P. heliodiscus (Z07) has a molecular weight of 2679 Da corresponding to the PLTX previously found in P. tuberculosa , P. toxica , P. caribaeorum and P. heliodiscus ( Béress et al., 1983; Deeds et al., 2011; Kimura & Hashimoto, 1973; Moore & Scheuer, 1971). The bishomo-PLTX isolated from P. heliodiscus (Z07) has a molecular weight of 2706 Da and is identical to the toxin identified in P. tuberculosa ( Uemura et al., 1985) . Finally, no PLTX or PLTX-like compounds were observed in P. mutuki , P. caribaeorum and P. grandis analysed in this study. Taken together, these results show a certain variability in both inter- and intraspecific toxicity in Palythoa . According to the most parsimonious scenario, phylogenetic patterns associated with comparative toxicity analysis suggest that the ability to produce and store a large amount of PLTX is likely a plesiomorphic character inherited from the last common ancestor of all Palythoa species that would have been lost independently in several lineages ( Fig. 6 ).

All toxic species investigated in this study were associated with Symbiodiniaceae belonging to the generalist genus Cladocopium which is the most species-rich, ecologically abundant, and broadly distributed genus within the Symbiodiniaceae ( LaJeunesse et al., 2018; Thornhill et al., 2014). While ITS2 of the rRNA gene remains the most popular marker used to infer the Symbiodiniaceae systematics ( Shi et al., 2021), the species genetic delineation within Cladocopium is generally very difficult due to a very low interspecies variability in these sequences ( Mizuyama et al., 2020; Reimer et al., 2017b; Thornhill et al., 2014). Considering ecological, geographic, reproductive, and genetic patterns, Cladocopium may consist of hundreds of species ( Thornhill et al., 2014). If so, there are likely distinct Cladocopium species present within toxic species of Palythoa , the identification of which may only be demonstrated with higher phylogenetic resolution markers ( LaJeunesse & Thornhill, 2011; Noda et al., 2017; Reimer et al., 2017b). To further describe the relationships between the Cladocopium strains and toxicity variability of their Palythoa hosts, it will be necessary to carry out studies based on alternative markers such as, for example, the plastid mini-circle non-coding region ( Mizuyama et al., 2020; Moore et al., 2003; Reimer et al., 2017b; Takishita et al., 2003). Meta-barcoding using NGS-based analyses should be also carried out for a more detailed picture of intra- and interspecific ITS diversity (see Hume et al., 2019).

Figures

Fig.6 Palytoxin (PLTX) contents in the genera Palythoa, Terrazoanthus, and Zoanthus determined in the present and previous studies. Red: high PLTX concentration (>300 µg/g wet). Orange: weak PLTX concentration. Green: no PLTX detected. Black: not determined. CAP: common ancestor of all Palythoa species. Palythoa tuberculosa (Esper, 1805) has not been used in the present study but Hirata et al. (1979) measured low concentration of PLTX (10 µg/g wet Paly-